The sarcoplasmic reticulum (SR) is a structure in cells that functions, among other things, as a specialized intracellular calcium (Ca2+) store. RyRs are channels in the SR, which open and close to regulate the release of Ca2+ from the SR into the intracellular cytoplasm of the cell. Release of Ca2+ into the cytoplasm from the SR increases cytoplasmic Ca2+ concentration. Open probability of RyRs refers to the likelihood that a RyR is open at any given moment, and therefore capable of releasing Ca2+ into the cytoplasm from the SR.
There are three types of RyR, all of which are highly homologous: RyR1, RyR2, and RyR3. RyR1 is found predominantly in skeletal muscle as well as other tissues, RyR2 is found predominantly in the heart as well as other tissues, and RyR3 is found in the brain as well as other tissues. The RyR is a tetramer. Part of the RyR complex is formed by four RyR polypeptides in association with four FK506 binding proteins (FKBPs) (calstabins), specifically FKBP12 (calstabin1) and FKBP12.6 (calstabin2). Calstabin1 binds to RyR1 and RyR3 while calstabin2 binds to RyR2. The calstabins bind to the RyR (one molecule per RyR subunit), stabilize the RyR function, facilitate coupled gating between neighboring RyRs and prevent abnormal activation (Ca2+ leak) of the channel by stabilizing the channel's closed state.
Ryanodine Receptor 2 and Cardiac Diseases
In cardiac striated muscle, RyR2 is the major Ca2+ release channel required for excitation-contraction (EC) coupling and muscle contraction. During EC coupling, depolarization of the cardiac-muscle cell membrane during phase zero of the action potential activates voltage-gated Ca2+ channels. Ca2+ influx through the open voltage-gated channels in turn initiates Ca2+ release from the SR via RyR2. This process is known as Ca2+-induced Ca2+ release. The RyR2-mediated Ca2+-induced Ca2+ release then activates the contractile proteins in the cardiac cell, resulting in cardiac muscle contraction.
Phosphorylation of RyR2 by protein kinase A (PKA) is an important part of the “fight or flight” response that increases cardiac EC coupling gain by augmenting the amount of Ca2+ released for a given trigger. This signaling pathway provides a mechanism by which activation of the sympathetic nervous system (SNS), in response to stress, results in increased cardiac output. Phosphorylation of RyR2 by PKA results in partial dissociation of calstabin2 from the channel, which in turn, leads to increased open probability, and increased Ca2+ release from the SR into the intracellular cytoplasm.
Heart failure (HF) is characterized by a sustained hyperadrenergic state in which serum catecholamine levels are chronically elevated. One consequence of this chronic hyperadrenergic state is persistent PKA hyperphosphorylation of RyR2, such that 3-4 out of the four Ser2808 in each homotetrameric RyR2 channel are chronically phosphorylated (Marx S O, et al. Cell, 2000; 101(4):365-376). In particular, chronic PKA hyperphosphorylation of RyR2 is associated with depletion of the channel-stabilization subunit calstabin2 from the RyR2 channel macromolecular complex. Depletion of calstabin results in a diastolic SR Ca2+“leak” from the RyR complex, which contributes to impaired contractility (Marx et al., 2000). Due to the activation of inward depolarizing currents, this diastolic SR Ca2+“leak” also is associated with fatal cardiac arrhythmias (Lehnart et al, J Clin Invest. 2008; 118(6):2230-2245). Indeed, mice engineered with RyR2 lacking the PKA phosphorylation site are protected from HF progression after myocardial infarction (MI) (Wehrens X H et al. Proc Natl Acad Sci USA. 2006; 103(3):511-518). In addition, chronic PKA hyperphosphorylation of RyR2 in HF is associated with remodeling of the RyR2 macromolecular complex that includes depletion of phosphatases (Marx et al. 2000) PP1 and PP2a (impairing dephosphorylation of Ser2808) and the cAMP-specific type 4 phosphodiesterase (PDE4D3) from the RyR2 complex. Depletion of PDE4D3 from the RyR2 complex causes sustained elevation of local cAMP levels (Lehnart S E, et al., Cell 2005; 123(1):25-35). Thus, diastolic SR Ca2+ leak contributes to HF progression and arrhythmias. Moreover, a recent report has demonstrated that RyR2-S2808D+/+ (aspartic acid replacing serine 2808) knock-in mice, that mimic constitutive PKA hyperphosphorylation of RyR2, show depletion of calstabin2 and leaky RyR2. RyR2-S2808D+/+ mice develop age-dependent cardiomyopathy, demonstrate elevated RyR2 oxidation and nitrosylation, a reduced SR Ca2+ store content, and increased diastolic SR Ca2+ leak. After myocardial infarction, RyR2-S2808D+/+ mice exhibit increased mortality compared with WT littermates. Treatment with S107, a 1,4-benzothiazepine derivative that stabilizes RyR2-calstabin2 interactions (WO 2007/024717), inhibited the RyR2-mediated diastolic SR Ca2+ leak and reduced HF progression in both WT and RyR2-S2808D+/+ mice (Shan et al., J Clin Invest. 2010 Dec. 1; 120(12):4375-87).
Moreover, RyR2 contains about 33 free thiol residues rendering it highly sensitive to the cellular redox state. Cysteine oxidation facilitates RyR opening and SR Ca2+ leak. Shan et al, 2010, demonstrated that oxidation and nitrosylation of RyR2 and dissociation of the stabilizing subunit calstabin2 from RyR2 induces SR Ca2+ leak.
Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an inherited disorder in individuals with structurally normal hearts. More than 50 distinct RyR2 mutations have been linked to CPVT. CPVT patients experience syncope and sudden cardiac death (SCD) from the toddler to adult ages, and by 35 years of age the mortality is up to 50%. Individuals with CPVT have ventricular arrhythmias when subjected to exercise, but do not develop arrhythmias at rest. CPVT-associated RyR2 mutations result in “leaky” RyR2 channels due to the decreased binding of the calstabin2 subunit (Lehnart et al., 2008). Mice heterozygous for the R2474S mutation in RyR2 (RyR2-R2474S mice) exhibit spontaneous generalized tonic-clonic seizures (which occurred in the absence of cardiac arrhythmias), exercise-induced ventricular arrhythmias, and SCD. Treatment with S107 enhanced the binding of calstabin2 to the mutant RyR2-R2474S channel, inhibited the channel leak, prevented cardiac arrhythmias and raised the seizure threshold (Lehnart et al., 2008).
Ryanodine Receptor 1 and Skeletal Muscle Diseases
Skeletal muscle contraction is activated by SR Ca2+ release via RyR1. Depolarization of the transverse (T)-tubule membrane activates the dihydropyridine receptor voltage sensor (Cav1.1) that in turn activates RyR1 channels via a direct protein-protein interaction causing the release of SR Ca2+ stores. Ca2+ binds to troponin C allowing actin-myosin cross-bridging to occur and sarcomere shortening.
In conditions of prolonged muscular stress (e.g., during marathon running) or in a disease such as heart failure, both of which are characterized by chronic activation of SNS, skeletal muscle function is impaired, possibly due to altered EC coupling. In particular, the amount of Ca2+ released from the SR during each contraction of the muscle is reduced, aberrant Ca2+ release events can occur, and Ca2+ reuptake is slowed (Reiken, S, et al. 2003. J. Cell Biol. 160:919-928). These observations suggest that the deleterious effects of chronic activation of the SNS on skeletal muscle might be due, at least in part, to defects in Ca2+ signaling.
The RyR1 macromolecular complex consists of a tetramer of the 560-kDa RyR1 subunit that forms a scaffold for proteins that regulate channel function including PKA and the phosphodiesterase 4D3 (PDE4D3), protein phosphatase 1 (PP1) and calstabin1. A-kinase anchor protein (mAKAP) targets PKA and PDE4D3 to RyR1, whereas spinophilin targets PP1 to the channel (Marx et al. 2000; Brillantes et al., Cell, 1994, 77, 513-523; Bellinger et al. J. Clin. Invest. 2008, 118, 445-53). The catalytic and regulatory subunits of PKA, PP1, and PDE4D3 regulate PKA-mediated phosphorylation of RyR1 at Ser2843 (Ser2844 in the mouse). It has been shown that PKA-mediated phosphorylation of RyR1 at Ser2844 increases the sensitivity of the channel to cytoplasmic Ca2+, reduces the binding affinity of calstabin1 for RyR1, and destabilizes the closed state of the channel (Reiken et al., 2003; Marx, S. O. et al., Science, 1998, 281:818-821). Calstabin1 concentrations in skeletal muscle are reported to be approximately 200 nM and that PKA phosphorylation of RyR1 reduces the binding affinity of calstabin1 for RyR1 from approximately 100-200 nM to more than 600 nM. Thus, under physiologic conditions, reduction in the binding affinity of calstabin1 for RyR1, resulting from PKA phosphorylation of RyR1 at Ser2843, is sufficient to substantially reduce the amount of calstabin1 present in the RyR1 complex. Chronic PKA hyperphosphorylation of RyR1 at Ser2843 (defined as PKA phosphorylation of 3 or 4 of the 4 PKA Ser2843 sites present in each RyR1 homotetramer) results in “leaky” channels (i.e., channels prone to opening at rest), which contribute to the skeletal muscle dysfunction that is associated with persistent hyperadrenergic states such as occurs in individuals with heart failure (Reiken et al., 2003).
Moreover, regulation of RyR1 by posttranslational modifications other than phosphorylation, such as by nitrosylation of free sulfhydryl groups on cysteine residues (S-nitrosylation), as well as channel oxidation, have been reported to increase RyR1 channel activity. S-nitrosylation and oxidation of RyR1 have each been shown to reduce calstabin1 binding to RyR1.
It was previously reported by Bellinger et al. (Proc. Natl. Acad. Sci. 2008, 105(6):2198-2002) that during extreme exercise in mice and humans, RyR1 is progressively PKA-hyperphosphorylated, S-nitrosylated and depleted of PDE4D3 and calstabin1, resulting in “leaky” channels that cause decreased exercise capacity in mice. Treatment with S107 prevented depletion of calstabin1 from the RyR1 complex, improved force generation and exercise capacity, and reduced Ca2+− dependent neutral protease calpain activity and plasma creatinine kinase levels.
Duchenne muscular dystrophy (DMD) is one of the leading lethal childhood genetic diseases. DMD is X-linked, affecting 1 in 3,500 male births and typically results in death by ˜30 y of age from respiratory or cardiac failure. Mutations in dystrophin associated with DMD lead to a complete loss of the dystrophin protein, thereby disrupting the link between the subsarcolemma cytoskeleton and the extracellular matrix. This link is essential for protecting and stabilizing the muscle against contraction induced injury. Currently, there is no cure for DMD and most treatments in the clinic are palliative. Emerging interventions in Phase I/II clinical trials are exon skipping, myostatin inhibition, and up-regulation of utrophin. However, problems with systemic delivery, sustaining exon skipping, and up-regulation of utrophin exist. In addition, in Phase I/II clinical trials, inactivation of myostatin to increase muscle size did not show improved muscle strength or function. Sarcolemmal instability due to mutations in dystrophin has a cascade effect. One major effect is increased cytosolic Ca2+ concentration, which leads to activation of Ca2+− dependent proteases (calpains). Another effect is inflammation and elevated iNOS activity, which can cause oxidation/nitrosylation of proteins, lipids, and DNA. DMD muscle pathology is progressive and far exceeds the instability of the sarcolemma. Thus the pathology is consistent with the instability of the sarcolemma increasing the susceptibility to further injury. It was recently demonstrated that excessive oxidation or nitrosylation of RyR1 can disrupt the interaction of calstabin1 with the RyR1 complex, leading to RyR1 leakiness and muscle weakness in a mouse model of muscular dystrophy (mdx) and that treatment with S107 improves indices of muscle function in this mouse model (Bellinger, A. et al. 2009, Nature Medicine, 15:325-330).
Age-related loss of muscle mass and force (sarcopenia) contributes to disability and increased mortality. Andersson, D. et al. (Cell Metab. 2011 Aug. 3; 14(2):196-207) reported that RyR1 from aged (24 months) mice is oxidized, cysteine-nitrosylated, and depleted of calstabin1, compared to RyR1 from younger (3-6 months) adults. This RyR1 channel complex remodeling resulted in “leaky” channels with increased open probability, leading to intracellular calcium leak in skeletal muscle. Treating aged mice with S107 stabilized binding of calstabin1 to RyR1, reduced intracellular calcium leak, decreased reactive oxygen species (ROS), and enhanced tetanic Ca2+ release, muscle-specific force, and exercise capacity.
PCT International patent publications WO 2005/094457, WO 2006/101496 and WO 2007/024717 disclose 1,4-benzothiazepine derivatives and their use in treating cardiac, skeletal muscular and cognitive disorders, among others.
PCT International patent publication WO 2008/060332 relates to the use of 1,4-benzothiazepine derivatives for treating muscle fatigue in subjects suffering from pathologies such as muscular dystrophy, or in subjects suffering from muscle fatigue as a result of sustained, prolonged and/or strenuous exercise, or chronic stress.
PCT International patent publication WO 2008/021432 relates to the use of 1,4-benzothiazepine derivatives for the treatment and/or prevention of diseases, disorders and conditions affecting the nervous system.
PCT International patent publication WO 2012/019076 relates to the use of 1,4-benzothiazepine derivatives for the treatment and/or prevention of cardiac ischemia/reperfusion injury. Fauconnier et al., Proc Natl Acad Sci USA, 2011, 108(32): 13258-63 reported that RyR leak mediated by caspase-8 activation leads to left ventricular injury after myocardial ischemia-reperfusion, and that treatment with S107 inhibited the SR Ca2+ leak, reduced ventricular arrhythmias, infarct size, and left ventricular remodeling at 15 days after reperfusion.
PCT International patent publication WO 2012/019071 relates to the use of 1,4-benzothiazepine derivatives for the treatment and/or prevention of sarcopenia.
PCT International patent publication WO 2012/037105 relates to the use of 1,4-benzothiazepine derivatives for the treatment and/or prevention of stress-induced neuronal disorders and diseases.
There is a need to identify new compounds effective for treating disorders and diseases associated with RyRs, including skeletal muscular and cardiac disorders and diseases. More particularly, a need remains to identify new agents that can be used to treat RyR-associated disorders by, for example, repairing the leak in RyR channels, and enhancing binding of calstabins to PKA-phosphorylated/oxidized/nitrosylated RyRs, and to mutant RyRs that otherwise have reduced affinity for, or do not bind to, calstabins.